Parallel Bioreactor System

ABSTRACT

According to the invention, there is provided a parallel bioreactor system, comprising: an oscillator for generating oscillating motion; a plurality of culture vessels mounted on the oscillator, wherein each culture vessel is provided with an inner cavity, the inner cavity comprises a cylindrical portion at the upper part and an inverted truncated conical bottom at the lower part, a cross section of the cylindrical portion is consistent with the cross section of the top of the inverted truncated conical bottom, and the bottom of the cylindrical portion is joined with the top of the inverted truncated conical bottom; disposable culture bags arranged in the inner cavities of the culture vessels and used for accommodating culture solution, wherein each disposable culture bag is provided with a multifunctional cover plate, and the multifunctional cover plate is connected to the top of the culture bag to seal the culture bag, and is provided with a plurality of connection holes leading to interior of the disposable culture bag; and a control system, wherein the control system controls the oscillating motion of the oscillator and parameters of the culture solution in the disposable culture bags.

FIELD OF THE INVENTION

The present invention provides a parallel bioreactor system and aculturing method using the parallel bioreactor system.

BACKGROUND OF THE INVENTION

The new generation of protein drugs represented by antibodies hasattracted more and more attention because of its advantages of goodtargeting, high curative effect, few side effects and the like, and ithas become the mainstream in the development of biotech drugsinternationally. Further, in recent years, as the avian flu, thefoot-and-mouth disease, the swine flu and other diseases have wreakedhavoc over the world, it is necessary to quickly develop and produce alarge number of urgently needed vaccines and related biological proteindrug products within a short period. Rapid production of theaforementioned protein drugs, antibodies and vaccine products relies onequipment really capable of implementing the large-scale culture ofanimal cells, i.e. bioreactors.

Bioreactors are a bridge connecting the laboratory technology researchand the scale production in the factory of the vaccines and the proteindrug.

In the past, the culturing mainly relates to E. coli and yeast systems,which have the advantages of being insensitive to shearing force, havinglarge oxygen consumption and being tolerant to pure oxygen. But membraneprotein or secretory protein cannot be expressed in prokaryotic cells,and they do not have functional activity similar to those of naturalantibodies, thereby being not the main direction of development.

At present, the sale of animal cell-expressed products accounts for 70%of biological drugs. Mammalian cells have become the most importantexpression/production system of modern biopharmacy. The only way torealize the large-scale cultivation of animal cells is the bioreactor.

A traditional reactor has three technical factors: oxygen transfer,mixing and control. At present, the main way for oxygen transfer isbubbling, airlift and stirring blade shearing. The main problem of thisway is that tension generated by the breakage of bubbles in bubbling andthe shearing force of a stirring blade damage the animal cells.

High throughput screening is one of the important technical means in thefield of life science and drug innovation, and its core is to get a lotof information through an experiment at a time and find valuableinformation therein. At the same time, stable and high-expression cellclones/cell strains and its optimized culturing strategies, culturingconditions and culturing technologies are also key factors for efficientand low-cost production of biological drugs.

Compared with traditional stainless steel bioreactors, disposablebioreactors have the advantages of simple operation, stable productionprocess, short preparation time between batches, high productionefficiency, no complicated pipelines and other auxiliary facilities, noneed of cleaning, disinfection and sterilization, low production cost,easy validation and the like, thereby having been developed andpopularized in the development and production of the modernbiotechnology drugs and having become the main trend in the developmentof bioreactors. The currently disclosed or commercially availabledisposable bioreactors mainly realize the transfer and mixing of allkinds of liquid, gases and other culture media in traditional manner ofstirring, bubbling or airlift, which has the shortcomings of highshearing force and large damage to sensitive cells or microorganisms,and thus is not conducive to high-density culture and production.Related prior art includes PCT application (WO 2013/186294) entitled“disposable bioreactor, top plate and related manufacturing method”, andthe technical solution thereof mainly relates to a disposable bioreactorthat can be applied to parallel culture of cells or microorganisms, atop plate thereof and a corresponding manufacturing method. Thebioreactor mainly realizes the transfer and mixing of the culture mediaby stirring and air introduction from the bottom, and a plurality ofindividual bioreactors are connected in parallel to achieve the parallelcontrol of the entire working process. The solution has the shortcomingsthat it cannot optimize dissolved oxygen levels for supporting thehigh-density production of cells, and will lead to differences inculture parameters between groups, thereby being unbeneficial for thehigh throughput screening of the cell clones and the accurateoptimization of the culturing process.

Therefore, there is a need for a multichannel biological cultureplatform which achieves high throughput screening and/or parallelculture of more than one type of samples while reducing the damage tothe sensitive cells or microorganisms.

SUMMARY OF THE INVENTION

According to a preferred embodiment of the present invention, theproblem that conventional reactors cannot achieve high throughputscreening of samples or cell clones and culture process optimization canbe solved. According to a preferred embodiment of the present invention,the problem that conventional high throughput shaking tables cannotachieve microenvironment control in a culture vessel can be solved.According to a preferred embodiment of the present invention, theshearing force is extremely small in the cell culturing, the dissolvedoxygen level is high, and the growth density of cells or microorganismssensitive to the shearing force can be effectively improved, thusproviding a great potential of improvement for process optimization andculture medium optimization. According to a preferred embodiment of thepresent invention, the parallel culture of multiple samples can becarried out at one time under the same external environment andconditions, and the microenvironment in each culture vessel iscontrollable, thus providing a preferred platform for high throughputscreening of the samples, and particularly the high throughput screeningof cell clones. According to a preferred embodiment of the presentinvention, the parallel culture of multiple candidate cell clones can becarried out at one time under the same external environment andconditions, and the microenvironment in each culture vessel iscontrollable, thus providing a preferred platform for the highthroughput screening of stable and high-expression cell clones andculture process optimization. According to a preferred embodiment of thepresent invention, the parallel culture of one same sample or cellstrain can be carried out under the same external environment andconditions, and the microenvironment in each culture vessel iscontrollable, thus providing a preferred platform for conductingsample/cell culture condition exploration and establishment, cultureprocess optimization, culture medium optimization and accumulation ofsmall amount of sample.

According to the first aspect of the present invention, there isprovided a parallel bioreactor system, comprising: an oscillator forgenerating oscillating motion; a plurality of culture vessels mounted onthe oscillator, wherein each culture vessel is provided with an innercavity, the inner cavity comprises a cylindrical portion at the upperpart and an inverted truncated conical bottom at the lower part, a crosssection of the cylindrical portion is consistent with the cross sectionof the top of the inverted truncated conical bottom, and the bottom ofthe cylindrical portion is joined with the top of the inverted truncatedconical bottom; disposable culture bags arranged in the inner cavitiesof the culture vessels and used for accommodating culture solution,wherein each disposable culture bag is provided with a multifunctionalcover plate, and the multifunctional cover plate is connected to the topof the culture bag to seal the culture bag, and is provided with aplurality of connection holes leading to interior of the disposableculture bag; and a control system, wherein the control system controlsthe oscillating motion of the oscillator and parameters of the culturesolution in the disposable culture bags.

Preferably, the disposable culture bag is a flexible culture bag.

Preferably, the flexible culture bag has a shape corresponding to thatof the inner cavity of the culture vessel when being unfolded.

Preferably, the outer shape of the culture vessel corresponds to theshape of the inner cavity, and comprises a cylindrical portion at theupper part and an inverted truncated conical bottom at the lower part.

Preferably, the oscillator comprises a support and a shaking plate, theshaking plate generates the oscillating motion relative to the support,and the culture vessels are mounted on the shaking plate.

Preferably, the shaking plate comprises a plurality of culture vesselholes, and each of the culture vessel holes has a shape matched with theouter shape of the culture vessels so as to at least partiallyaccommodate one culture vessel.

Preferably, the culture vessel hole has an inverted truncated conicalbottom.

Preferably, the culture holes are structurally distributed in arectangular array or an annular array.

Preferably, 16 culture vessels are provided and are evenly mounted onthe shaking plate in a matrix form of 4 rows and 4 columns.

Preferably, the oscillator is provided with a motor, a main transmissioneccentric shaft and supporting eccentric shafts, the main transmissioneccentric shaft and the supporting eccentric shafts are connectedbetween the support and the shaking plate by bearings, the motor drivesthe main transmission eccentric shaft and thus drives the shaking plateto carry out rotary reciprocating horizontal oscillating motionaccording to a set amplitude. Preferably, the oscillator comprises foursupporting eccentric shafts, which are evenly distributed on the bottomof the oscillator, a balancing weight is mounted on each supportingeccentric shaft, and the balancing weight forms an angle of 180° withthe eccentric direction to balance a centrifugal force generated by aload in an oscillating process of the oscillator. Preferably, adiameter-height ratio of the inverted truncated conical bottom isgreater than 1:1, and the taper angle of the inverted truncated conicalbottom is within a range of 30°-70°.

Preferably, each connection hole of the multifunctional cover plate is athread interface of a unified standard.

Preferably, the connection holes of the multifunctional cover plate aresuitable for being connected with a detection electrode or a conduit.

Preferably, 6-12 connection holes are provided.

Preferably, the system further comprising a perfusion system, whereinthe perfusion system comprises a bracket having two guide posts and aperfusion type culture bag vessel fixed between the two guide posts, andlifting adjustment buttons are arranged at lower ends of the guideposts.

Preferably, the perfusion type culture bag vessel is connected with theculture vessel through pipelines and the connection holes of themultifunctional cover plate to form an outer circulation type perfusionculture mode.

Preferably, the control system comprises a manual control mode and anautomatic control mode.

Preferably, the control system monitors and controls one or more of thefollowing parameters in the disposable culture bag disposed in theculture vessel: liquid level, temperature, pH value and dissolved oxygenlevel.

Preferably, the control system can independently monitor and controleach disposable culture bag.

Preferably, the control system comprises a master control console and aplurality of reaction controllers; the master control console controlsthe oscillator and is connected to the plurality of reaction controllersto receive data from the plurality of reaction controllers and send acontrol instruction to the plurality of reaction controllers; and eachreaction controller is connected to the corresponding culture vessel toreceive the parameters from the culture vessel and carry out relatedoperations on the culture vessel.

Preferably, the plurality of culture vessels are divided into at leasttwo groups, and identical or different cells or microorganisms arecultured in each group of culture vessels.

Preferably, the control system controls the groups of culture vessels tohave different culture parameters therebetween.

According to the second aspect of the invention, there is provided aculture method for culturing cells and/or microorganisms by using theparallel bioreactor system of the first aspect of the invention,comprising: independently monitoring and controlling each culture vesselthrough the control system.

Preferably, the method comprises dividing the plurality of culturevessels into at least two groups, and culturing identical or differentcells or microorganisms in each group of culture vessels.

Preferably, the method comprises controlling the groups of culturevessels to have different culture parameters therebetween.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate embodiments of the presentinvention in an exemplary way without any limitation.

FIG. 1 shows a parallel bioreactor system according to an embodiment ofthe present invention.

FIG. 2 shows a disposable culture bag according to an embodiment of thepresent invention.

FIG. 3 shows a multifunctional cover plate according to an embodiment ofthe present invention.

FIG. 4 shows a perspective view of a culture vessel, a disposableculture bag and a multifunctional cover plate that are assembledaccording to an embodiment of the present invention.

FIG. 5 shows multiple culture vessels mounted on a shaking plateaccording to an embodiment of the present invention.

FIG. 6 shows a schematic diagram of an oscillator according to anembodiment of the present invention.

FIG. 7 shows exemplary layout of a control system according to thepresent invention.

FIG. 8 shows an exemplary working principle of a perfusion systemaccording to the present invention.

FIG. 9 shows a schematic connection diagram between a culture vessel anda reaction controller and the like according to an exemplary embodimentof the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Hereinafter, embodiments of the present invention will be described withreference to the accompany drawings. The following description is merelyexemplary and is not intended to limit the protection scope of thepresent invention.

A parallel bioreactor system according to an embodiment of the presentinvention is based on a non-bubbling oxygen transfer mechanism, whereina plurality of culture vessels having inverted truncated conical(inverted frustum of a cone) inner cavities are placed on the sameplatform (shaking plate or shaking table), and the platform is driven byan oscillator to achieve eccentric oscillating operation. In someembodiments, the parameter control of each culture vessel can becontrolled by an independent CPU and actuator(s), records, reports andother data management are relatively independent, and the pH value,dissolved oxygen, nutrients and other parameters in cell culturemicroenvironments can be accurately regulated and controlled. Adisposable culture bag with a matching shape and structure is providedin the culture vessel and is used once being unpacked, so that crosscontamination can be avoided, the inter-batch treatment period can beshortened, no washing, disinfection or verification is needed, thusgreatly improving the working efficiency. Due to the shape of the innercavity and the oscillating motion of the culture vessel, the shearingforce in the entire cell culture process is extremely small, thedissolved oxygen efficiency is high, the growth density of cells ormicroorganisms sensitive to the shearing force can be effectivelyimproved, thus providing a great potential improvement for processoptimization and culture medium optimization. At the same time, due tothe high-level dissolved oxygen efficiency, the oxygen toxicity on thecells by pure oxygen during the high-density growth of the cells ormicroorganisms can be avoided, and the problems that conventionalreactors cannot achieve high throughput screening and that conventionalhigh throughput shaking tables cannot achieve microenvironment controlin the culture vessel can be solved. The parallel bioreactor systemaccording to the present invention can achieve parallel culture of asingle variety of sample, and can also be used for culturing multipledifferent varieties of samples at the same time on one platform, andthus the parallel bioreactor system can be widely applied tohigh-expression cell clone screening, culture condition exploration,culture process optimization, culture medium optimization and other newproduct development processes of biological pharmacy.

The parallel bioreactor system according to the present invention mainlyincludes an oscillator, a plurality of culture vessels and a controlsystem. FIG. 1 shows a parallel bioreactor system according to anembodiment of the present invention, including a plurality of culturevessels 101, an oscillator 102 and a control system. For example, thecontrol system may include a master control console 104 and a pluralityof reaction controllers 103.

For example, the master control console 104 can be a computer (an uppercomputer), which operates a control program and is provided with aninput interface to receive the input of an operator. Preferably, thenumber of the reaction controllers 103 can be the same as the number ofthe culture vessels 101, and each reaction controller 103 is separatelyconnected to and/or controls sensor(s) and actuator(s) associated withone corresponding culture vessel 101. Therefore, the parallel bioreactorsystem according to the present invention can independently controlrelated operations of each culture vessel, which is particularlyadvantageous in the case that multiple different varieties of cellsand/or microorganisms are cultured at the same time. Common parameters(e.g., a rotating speed of the oscillator or the like) of the culturevessels can be collectively controlled by the master control console104. For example, the plurality of culture vessels can be divided intoat least two groups, and different cells or microorganisms are culturedbetween each group of culture vessels. For example, the plurality ofculture vessels are divided into a first group and a second group, firstcells or microorganisms are cultured in the first group, and secondcells or microorganisms are cultured in the second group.

The plurality of culture vessels are arranged on the same shaking plateand the parameters in each vessel can be independently controlled,therefore the system according to the present invention can carry outparallel culture of multiple candidate cell clones at one time under thesame external environment and conditions, and the microenvironment ineach culture vessel is controllable, thus providing a preferred platformfor high throughput screening of stable and high-expression cell clonesand culture process optimization. Similarly, the plurality of culturevessels are arranged on the same shaking plate and the parameters ineach vessel can be independently controlled, therefore the systemaccording to the present invention can carry out the parallel culture ofone same sample or cell strain under the same external environment andconditions, and the microenvironment in each culture vessel iscontrollable, thus providing a preferred platform for sample/cellculture condition exploration and establishment, culture processoptimization, culture medium optimization and accumulation of smallamount of sample.

FIG. 9 shows a schematic connection diagram between a culture vessel anda corresponding reaction controller and the like in an adherent culturemode (or attachment culture mode) according to an exemplary embodimentof the present invention. The connection between the culture vessel andthe reaction controller thereof and the connection between the culturevessel, the reaction controller and other portions can be properly setaccording to culture needs.

FIG. 2 shows a disposable culture bag according to an embodiment of thepresent invention. FIG. 3 shows a multifunctional cover plate accordingto an embodiment of the present invention. FIG. 4 shows a perspectiveview of a culture vessel, a disposable culture bag and a multifunctionalcover plate that are assembled according to an embodiment of the presentinvention.

An inner cavity or a culture chamber is defined by an inner surface of aculture vessel 101 according to the present invention. As shown in FIG.2, the inner cavity includes a cylindrical portion at an upper part andan inverted truncated conical bottom (or an inverted frustum-shapedbottom) at a lower part, a cross section of the cylindrical portion isconsistent with the cross section of the top of the inverted truncatedconical bottom, and the bottom of the cylindrical portion is joined withthe top of the inverted truncated conical bottom. The cylindricalportion and the inverted truncated conical bottom are joined as anintegral whole and have a common rotation axis, as shown in the figure.

The shape of an outer surface (or outer appearance) of the culturevessel 101 is not limited. Preferably, as shown in the figure, the outerappearance of the culture vessel corresponds to the shape of the innercavity and includes a cylindrical portion at the upper part and aninverted truncated conical bottom at the lower part. In otherembodiments, the shape of the outer surface of the culture vessel 101can be a cylinder, a cone and the like, as long as it can beconveniently manufactured, stored, transported and mounted. In someembodiments, a portion used for fixing the culture vessel 101 to theoscillator can be arranged at the outside of the culture vessel 101, forexample, a flange for passing through a bolt, etc.

Preferably, the culture vessel 101 is at least partially inserted in aculture vessel hole (or mounting hole) formed in the shaking plate, theshape of the culture vessel hole is matched with the outer shape of theculture vessel 101, so that the culture vessel 101 can be stably mountedon the shaking plate. The culture vessel 101 can be completely insertedin the culture vessel hole. Alternatively, the culture vessel ispartially inserted in the culture vessel hole. For example, the culturevessel hole includes a cylindrical portion at the upper part and aninverted truncated conical bottom at the lower part, but the height ofthe cylindrical portion is less than the height of the cylindricalportion of the culture vessel. Or, the culture vessel hole can onlyinclude the inverted truncated conical bottom.

A disposable culture bag is arranged in a corresponding culture vesselaccording to the present invention. Preferably, the disposable culturebag is a flexible culture bag. In other embodiments, the disposableculture bag can also be made of a hard material. The disposable culturebag has a shape consistent with that of the inner cavity of the culturevessel (i.e., the appearance and the inner shape thereof are consistentwith the shape of the inner cavity of the culture vessel). For example,when the disposable culture bag is the flexible culture bag, it has asmaller wall thickness, and the outer shape and the inner shape whenunfolded are consistent with that of the inner cavity of the culturevessel.

That is to say, the unfolded disposable culture bag also includes acylindrical portion at the upper part and an inverted truncated conicalbottom (or an inverted frustum-shaped bottom) at the lower part, thecross section of the cylindrical portion is consistent with the crosssection of the top of the inverted truncated conical bottom, and thebottom of the cylindrical portion is joined with the top of the invertedtruncated conical bottom. The cylindrical portion and the invertedtruncated conical bottom are joined as an integral whole and have acommon rotation axis.

A diameter-height ratio of the inverted truncated conical bottom of theculture vessel of the present invention is greater than 1:1, so that aratio of the superficial area of a culture medium in the culture vesselto the volume of the culture medium is greater than 0.14 cm²/cm³.

The culture vessel of the present invention has a flat bottom of aninverted truncated cone (the inverted truncated conical bottom), whichis conducive to guiding the culture medium into the culture vessel, andthe superficial area of the culture medium is obviously large, which isbeneficial for the culture medium to contact an oxygen-containing gas inthe vessel and also beneficial for the escape of the gas in the culturemedium. Under the drive of the shaking plate of the oscillator, theculture medium can circularly brush the inner surface of the vessel toform a thin culture medium layer in a larger range, so as to furtherexpand the superficial area of the culture medium, increase thethroughput, improve the mixing, generate no shearing force or extremelysmall shearing force and generate extremely small mechanical stress.

According to the culture vessel of the present invention, the air canenter into the body of the culture vessel.

According to the culture vessel of the present invention, preferably,the taper angle of the cone body of the inverted truncated cone is30°-70°. Since the angle of such range is adopted, the inoculationvolume of seed cells or microorganisms in the vessel can be furtherreduced, so that a larger area of the culture medium is acquired in aculture process, and meanwhile a better mixing effect is obtained.Further, the cost can be further reduced, and higher applicationfriendliness is achieved.

Preferably, the volume of each culture vessel according to the presentinvention is in 0.3L-5 L, the parameter control of each culture vesselcan be controlled by independent control channels, and records, reportsand other data management are relatively independent. FIG. 5 shows aplurality of culture vessels mounted on the shaking plate according toan embodiment of the present invention. The parallel bioreactor systemaccording to the present invention can include up to 50 culture vessels.In FIG. 5, 16 culture vessels are provided and are mounted on theshaking plate in a rectangular array. According to other embodiments,the culture vessels can also be mounted on the shaking plate in anannular array. The plurality of culture vessels in the present inventionhave the same structures and features and are arranged on the shakingplate of the same oscillator, and thus the same external workingparameters can be obtained.

Preferably, the culture vessel according to the present invention can becomposed of glass, metal and other materials.

In a cell (or microorganism) culture process, various parameters in acell growth environment must be monitored in real time, and the cultureenvironment or conditions must be correspondingly monitored and changedaccording to the changes of the parameters. The traditional methods formonitoring the parameters of the cell culture environment is generallycarried out by directly taking out cell culture solution to performoffline monitoring, or by fixedly placing each detection electrode at acertain fixed position in the cell culture vessel. Meanwhile, culturebag interfaces are different and cannot be universally applied todifferent types of cell culture, resulting in higher manufacturing andusing costs.

According to the present invention, a multifunctional cover plate can bearranged at the top of the disposable culture bag. Preferably, thedisposable culture bag and the multifunctional cover plate areintegrated. For example, the culture bag is directly welded on the coverplate in a physical welding manner to constitute an integral component.Since the shape of the disposable culture bag after being inflated orfilled with the culture solution (being unfolded) is consistent withthat of the culture vessel, after being inflated or filled with theculture solution by a pipeline, the disposable culture bag can be firmlyabutted to the culture vessel under the action of the gravity. Otherrestraining or fixing devices can also be set to keep the disposableculture bag if necessary.

Preferably, 6-12 connection holes are collectively formed in the coverplate, and the connection holes lead to the inside of the disposableculture bag and can be sealed by threads with good gas tightness.According to the needs of cell culture, any connection holes can be usedfor connecting to the detection electrodes to online monitor thetemperature, the dissolved oxygen, the pH and other environmentalparameters in the cell culture process. Or, conduits are connectedthrough the connection holes to carry out cell culture inoculation,culture solution adding, sampling, recycling, harvest, gas change andother operations, so as to further optimize the culture conditions andimprove the cell culture density. Meanwhile, the connection holes in themultifunctional cover plate that can be applied to various disposableculture bags may be thread interfaces using a unified standard, so thatthe gas tightness is good, and the connection holes can be used flexiblyaccording to the needs of the cell culture. Unnecessary connection holesin a specific culture process can be sealed easily.

As shown in FIG. 3, 11 connection holes (interfaces) are formed in themultifunctional cover plate according to a preferred embodiment of thepresent invention, and can be sealed by threads with good gas tightness.The 11 connection holes are respectively standby holes 1-3, a samplingport, a temperature electrode port, a liquid infusion port, a liquidcollection port, a DO (dissolved oxygen) electrode port, a pH electrodeport, a gas inlet and a gas outlet, which can be flexibly used accordingto the needs of the cell culture and can be applied to various types ofdisposable culture bags.

FIG. 4 shows a perspective view of a culture vessel, a disposableculture bag and a multifunctional cover plate that are assembledaccording to an embodiment of the present invention. In the embodiment,the outer appearance of the culture vessel corresponds to the shape ofthe inner cavity and includes the cylindrical portion at the upper partand the inverted truncated conical bottom at the lower part. The outerappearance and the shape of the inner volume of the unfolded disposableculture bag are consistent with the shape of the inner cavity of theculture vessel. The multifunctional cover plate is arranged at the topof the disposable culture bag, and the multifunctional cover plate isprovided with a plurality of connection holes.

FIG. 6 shows a schematic diagram of an oscillator according to anembodiment of the present invention.

The oscillator according to present invention mainly includes a supportand a shaking plate, and the shaking plate can generate oscillatingmotion relative to the support. In one embodiment, the oscillatorfurther includes a motor, a main transmission eccentric shaft andsupporting eccentric shafts. The main transmission eccentric shaft andthe supporting eccentric shafts are fixed between the support and theshaking plate by bearings, and the motor is connected to the maintransmission eccentric shaft through a belt. Four supporting eccentricshafts of the oscillator are evenly distributed at the bottom of theoscillator, and a balancing weight is mounted on each of the supportingeccentric shaft to play a support role. The balancing weight forms anangle of 180° with the eccentric direction to balance a centrifugalforce generated by a load in an oscillating process of the oscillator.The shaking plate can carry out eccentric rotation about a verticalrotating center in the horizontal direction or plane, and since theshaking plate is supported by the four supporting eccentric shafts, theshaking plate can bear a very large load. Meanwhile, in the oscillatingprocess of the oscillator, the supporting eccentric shafts are providedwith the balancing weights to balance the load, and thus the oscillatorbody will generate no displacement. Under the drive of the motor, theshaking plate of the oscillator carries out rotary reciprocatinghorizontal oscillating motion, namely horizontal eccentric rotation,according to a set amplitude.

As shown in FIG. 6, according to an embodiment of the present invention,the oscillator includes a support 1, bearings 2, a motor 3, a shakingplate 7, a main transmission eccentric shaft 6, supporting eccentricshafts 5 and balancing weights 4, wherein the supporting eccentricshafts 5 and the main transmission eccentric shaft are fixed on thesupport 1 by the bearings 2 and are connected to the shaking plate bythe bearings, and the motor 3 is connected with the main transmissioneccentric shaft 6 by a belt 8 to drive the main transmission eccentricshaft 6 to rotate. Since the main transmission eccentric shaft 6 isfixed on the support 1, the eccentric portion thereof is connected tothe shaking plate 7 as shown in the figure, and therefore when the maintransmission eccentric shaft 6 rotates, it drives the shaking plate 7 tocarry out horizontal rotary oscillation about a vertical direction. FIG.7 shows an exemplary layout of a control system according to the presentinvention.

The control system may include a main controller (e.g., a computer), acontrol circuit and an automatic control program. For example, thecontrol circuit can include a variety of controllers, sensors, drives,connection pipelines and circuits. Usually, the control circuit caninclude a PLC (programmable logic controller), a touch screen, anelectric control board, a peristaltic pump, a blower, a solenoid valve,a liquid level pump, an air pump, an electronic ruler, a rotating speedsensor, a DO (dissolved oxygen) transmitter, a PH transmitter, a Telectrode, a frequency converter, an SF drive (servo driver), a relay, aheating film, etc. The electric control board is respectively connectedwith the peristaltic pump, the blower, the solenoid valve, the liquidlevel pump and the air pump, a PLC is respectively connected with thetouch screen, the electric control board, the electronic ruler, therotating speed sensor, the DO transmitter, the PH transmitter, the Telectrode, the frequency converter, the SF driver and the relay, whereinthe frequency converter controls the oscillator, the SF drive controls aperfusion lifting frame, and the relay controls the heating film.

The arrangement and connection of the control circuit according to thepresent invention can be adjusted according to the needs. For example,the sensors in the culture vessel can be connected through theconnection holes of the multifunctional cover plate to obtaincorresponding data. A culture solution input pipeline and the like canbe connected to the culture vessel through the connection holes of themultifunctional cover plate, and the solenoid valve is arranged at aproper position to control the on/off of the pipelines. FIG. 3 shows anexemplary arrangement of the connection holes in the multifunctionalcover plate, and corresponding control circuit arrangement can be seen.The control system according to the present invention can separatelycontrol each of the plurality of culture vessel, so related arrangementsat each cover plate can be different from each other.

The control system has two operation modes, i.e. a manual control modeand an automatic control mode.

In the manual control mode, corresponding operations of the controlsystem can be controlled through the upper computer, and the parametersin the culture vessel of the system are adjusted, so that theenvironment of the culture vessels is suitable for the culture needs ofcells or microorganisms.

In the automatic control mode, the necessary parameters can be presetaccording to the culture needs of cells or microorganisms, and thesystem automatically adjusts the environment in the culture vessel in anautomatic adjustment mode to meet the environment necessary for the freeculture and growth of cultures.

According to the embodiment of the present invention, the automaticcontrol of some parameters is described as follows.

Generally, calibration operation is needed before a temperature, a pHvalue or a dissolved oxygen signal is collected for the first time. Atthe same time, a harvest pump and a liquid infusion pump also need to becalibrated in order to make the volume of liquid in the culture vesselapproximately maintain a balance.

1. Temperature Control

The temperature signal is converted by a PT100 transducer into a 4-20 mAcurrent signal to enter an analog input channel of the control system.The control system automatically converts the 4-20 mA current signalinto a corresponding temperature value. The control system compares thecollected temperature value with a system set value. If the collectedvalue is much smaller than the set value, a temperature heating valve isnormally open. If a sampling value is less than the set value within acertain range, the control system carries out PMW mode control andperiodically controls the on/off of an output heating valve. If thesampling value is greater than the set value, the heating valve isdisconnected.

2. PH Value Control

A pH sensor converts the collected signal into the 4-20 mA currentsignal by the transducer and conveys the same to the analog inputchannel of the control system. The control system automatically convertsthe 4-20 mA current signal into a corresponding pH value.

1) Operation on a PH Upper Limit

If the collected value is far greater than the set value, then a CO2valve is normally open. If the sampling value is greater than the setvalue within a certain range, the control system carries out the PWMmode control and periodically controls the on/off of an output CO2valve. If the sampling value is less than the set value, then the CO2valve is disconnected.

2) Operation on a PH Lower Limit

If the collected value is far less than the set value, an alkali addingpump valve is normally open. If the sampling value is less than the setvalue within a certain range, the control system carries out the PWMmode control and periodically controls the on/off of an output alkaliadding pump valve. If the sampling value is greater than the set value,then the alkali adding pump valve is disconnected.

3) The sampling value between the upper limit and the lower limit of thepH

Neither of the CO2 valve and the alkali adding pump valve is opened.

3. Dissolved Oxygen Control

An oxygen content sensor converts the collected signal into the 4-20 mAcurrent signal and conveys the same to the analog input channel of thecontrol system. The control system automatically converts the currentsignal into a corresponding dissolved oxygen value.

1) Operation on a Dissolved Oxygen Upper Limit

If the collected value is far greater than the set value, then a N2(nitrogen) filling valve is normally open. If the sampling value isgreater than the set value within a certain range, the control systemcarries out the PWM mode control and periodically controls the on/off ofan N2 output valve. If the sampling value is less than the set value,then the N2 valve is disconnected.

2) Operation on a Dissolved Oxygen Lower Limit If the collected value isfar less than the set value, an O2 (oxygen) adding valve is normallyopen. If the sampling value is less than the set value within a certainrange, the control system carries out the PWM mode control andperiodically controls the on/off of an O2 output valve. If the samplingvalue is greater than the set value, then the O2 valve is disconnected.

3) If the Sampling Value is Between the Upper Limit and the Lower Limitof the Dissolved Oxygen

Neither of the N2 valve and the O2 valve is opened.

4. Control of Harvest and Liquid Infusion of the System

In the cell culture process, the culture solution in the culture vesselis always consumed. The system needs to add a certain amount of culturesolution regularly to the culture vessel to ensure the growth of thecells. In order to make the amount of liquid contained in the culturevessel basically unchanged, the amount of harvest and the amount ofliquid infusion need to basically reach a balance, and this goal can beachieved by harvesting the culture solution by a system harvest pumpwhile carrying out liquid infusion.

By setting the corresponding total liquid infusion amount or totalharvest amount and the corresponding total time amount at one time, thesystem automatically calculates the time required for liquid infusion orharvest of the system within each minute. Therefore, the work of thecorresponding liquid infusion pump or harvest pump can be controlled bythe aforementioned PWM mode.

5. Setting and Control of a Shaking Table Rotating Speed

The system converts a given shaking table rotating speed signal tooutput through the analog output channel, and drives the motor to rotateaccording to the given rotating speed. In an operation process, theactual rotating speed of the system is detected by the input of aphotoelectric detection switch. If the actual rotating speed deviatesfrom the given rotating speed, the system will automatically adjust therotating speed, so that the actual rotating speed is matched with theset rotating speed.

6. Alarm Output of the System

After upper and lower alarm limits of the temperature, the pH, thedissolved oxygen, the shaking table rotating speed and the like are set,if the actual parameter value of the system is greater than the upperalarm limit or less than the lower alarm limit, a system alarm signal istriggered, and a buzzer alarms. Meanwhile, the reason for triggering thealarm signal is displayed on the upper computer, so that the operatorcan conveniently check it.

7. Automatic Control Program of a Perfusion Servo System in the Casethat the Perfusion System is Adopted (which will be described withReference to FIG. 8)

a) Liquid Level Control of the Perfusion System

The liquid level of the perfusion system is controlled by a high and lowlevel photoelectric detection switch, and when the liquid level of thesystem is between the high level and the low level, the motor of theperfusion system does not work. When a low level signal is detected, theperfusion motor infuses liquid so that the liquid level enters betweenthe high level and the low level and continues operating for a certainperiod of time; and when a high level signal is detected, the perfusionmotor discharges the liquid so that the liquid level enters between thehigh level and the low level and continues operating for a certainperiod of time. Therefore, the liquid level of the system is alwayslocated in a relatively stable liquid level area. The startups ofperfusion liquid infusion and perfusion liquid discharge areinterlocked.

b) Servo Motor Control of the Perfusion System

Zero point calibration needs to be carried out in the perfusion systemat first, and after a lifting speed and a target position are set, anexecution button is pressed, and the perfusion system moves towards thetarget position.

In the parallel bioreactor system according to the present invention,the plurality of culture vessels are arranged on the same oscillatorshaking plate to carry out parallel culture of a single variety and canalso be used for culturing multiple different varieties at the same timeon one platform. Since the shaking plate of the oscillator collectivelydrives the culture vessels with the inverted truncated conical bottomsto shake, sufficient dissolved oxygen and a unified culture rate can beprovided to support the growth of high-density cells. Accordingly, thehigh throughput screening and culture process optimization ofhigh-expression cell clones can be achieved under high-density cellculture conditions. The entire culture process is high in efficiency,easy to operate and low in cost, and thus can be widely applied tohigh-expression cell clone screening, culture condition exploration,culture process optimization, culture medium optimization, seed chainamplification and other new product development processes of biologicalpharmacy.

According to the aforementioned embodiment of the present invention,since the plurality of culture vessels are provided and each culturevessel can be independently controlled, the problem that conventionalreactors cannot achieve high throughput screening of samples or cellclones culture process optimization can be solved. According to theaforementioned embodiment of the present invention, since each culturevessel can be independently controlled, the problem that conventionalhigh throughput shaking tables cannot achieve microenvironment controlin the culture vessel can be solved.

According to the aforementioned embodiment of the present invention,since the shapes of the inner cavity of the culture vessel and thedisposable culture bag are inverted truncated cones, the shearing forceis extremely small in the cell culture, the dissolved oxygen level ishigh, and the growth density of cells or microorganisms sensitive to theshearing force can be effectively improved, thus providing a greatpotential of improvement for process optimization and culture mediumoptimization. According to the aforementioned embodiment of the presentinvention, since the plurality of culture vessels are arranged on thesame shaking plate and the parameters in each culture vessel can beindependently controlled, the parallel culture of multiple samples canbe carried out at one time under the same external environment andconditions, and the microenvironment in each culture vessel iscontrollable, thus providing a preferred platform for high throughputscreening of the samples, and particularly the high throughput screeningof cell clones. According to the aforementioned embodiment of thepresent invention, the parallel culture of multiple candidate cellclones can be carried out at one time under the same externalenvironment and conditions, and the microenvironment in each culturevessel is controllable, thus providing a preferred platform for the highthroughput screening of stable and high-expression cell clones andculture process optimization. According to the aforementioned embodimentof the present invention, the parallel culture of one same sample orcell strain can be carried out under the same external environment andconditions, and the microenvironment in each culture vessel iscontrollable, and thus providing a preferred platform for sample/cellculture condition exploration and establishment, culture processoptimization, culture medium optimization and accumulation of smallamount of sample.

FIG. 8 shows an exemplary working principle of a perfusion systemaccording to the present invention.

As a further solution, a perfusion system used for culturing specificcells or microorganisms is mounted on the parallel bioreactor systemaccording to the present invention. The perfusion system mainly includesa bracket, bracket lifting adjusting buttons, a peristaltic pump, guideposts, a manual adjusting wheel and a perfusion culture bag vessel. Theperfusion culture bag vessel is fixed between two guide posts on thebracket through fixing frames, the bracket lifting adjusting button isarranged at the lower ends of the guide posts, the peristaltic pump isfixed in a cabinet, and the manual adjusting wheel is fixed on a movableplate on the guide posts.

When the perfusion system is used for culturing specific cells ormicroorganisms, the culture vessel provides the dissolved oxygen and theculture solution, and the pH, the temperature and other conditionssuitable for cell growth are adjusted and controlled online. The culturesolution is injected into the perfusion culture bag at a controllableflow rate and flows back into the culture vessel at a controllable flowrate under the action of the gravity, so as to form an outer circulationtype perfusion culture mode. A culture carrier is fixed in the perfusionculture bag, and adherent cells or microorganisms are adsorbed on theculture carrier to grow. The culture solution flows by the carrier toprovide nutrients necessary for the cultures and take away metabolites,so as to form a stable fluid environment on the surrounding of thecultures and provide a three-dimensional structure of growth, interflowand cell clusters formation, so as to achieve the purpose ofhigh-density cell culture.

The present invention is not limited to the aforementioned specificdescription, and any modifications that are conceivable to those skilledin the art on the basis of the aforementioned description fall withinthe scope of the present invention.

1. A parallel bioreactor system, comprising: an oscillator forgenerating oscillating motion; a plurality of culture vessels mounted onthe oscillator, wherein each culture vessel is provided with an innercavity, the inner cavity comprises a cylindrical portion at the upperpart and an inverted truncated conical bottom at the lower part, a crosssection of the cylindrical portion is consistent with the cross sectionof the top of the inverted truncated conical bottom, and the bottom ofthe cylindrical portion is joined with the top of the inverted truncatedconical bottom; disposable culture bags arranged in the inner cavitiesof the culture vessels and used for accommodating culture solution,wherein each disposable culture bag is provided with a multifunctionalcover plate, and the multifunctional cover plate is connected to the topof the culture bag to seal the culture bag, and is provided with aplurality of connection holes leading to interior of the disposableculture bag; and a control system, wherein the control system controlsthe oscillating motion of the oscillator and parameters of the culturesolution in the disposable culture bags.
 2. The parallel bioreactorsystem of claim 1, wherein the disposable culture bag is a flexibleculture bag.
 3. The parallel bioreactor system of claim 2, wherein theflexible culture bag has a shape corresponding to that of the innercavity of the culture vessel when being unfolded.
 4. The parallelbioreactor system of claim 1, wherein the outer shape of the culturevessel corresponds to the shape of the inner cavity, and comprises acylindrical portion at the upper part and an inverted truncated conicalbottom at the lower part.
 5. The parallel bioreactor system of claim 1,wherein the oscillator comprises a support and a shaking plate, theshaking plate generates the oscillating motion relative to the support,and the culture vessels are mounted on the shaking plate.
 6. Theparallel bioreactor system of claim 5, wherein the shaking platecomprises a plurality of culture vessel holes, and each of the culturevessel holes has a shape matched with the outer shape of the culturevessels so as to at least partially accommodate one culture vessel. 7.The parallel bioreactor system of claim 6, wherein the culture vesselhole has an inverted truncated conical bottom.
 8. The parallelbioreactor system of claim 5, wherein the oscillator is provided with amotor, a main transmission eccentric shaft and supporting eccentricshafts, the main transmission eccentric shaft and the supportingeccentric shafts are connected between the support and the shaking plateby bearings, the motor drives the main transmission eccentric shaft andthus drives the shaking plate to carry out rotary reciprocatinghorizontal oscillating motion according to a set amplitude.
 9. Theparallel bioreactor system of claim 8, wherein the oscillator comprisesfour supporting eccentric shafts, which are evenly distributed on thebottom of the oscillator, a balancing weight is mounted on eachsupporting eccentric shaft, and the balancing weight forms an angle of180° with the eccentric direction to balance a centrifugal forcegenerated by a load in an oscillating process of the oscillator.
 10. Theparallel bioreactor system of claim 1, wherein a diameter-height ratioof the inverted truncated conical bottom is greater than 1:1, and thetaper angle of the inverted truncated conical bottom is within a rangeof 30°-70°.
 11. The parallel bioreactor system of claim 1, wherein theconnection holes of the multifunctional cover plate are suitable forbeing connected with a detection electrode or a conduit.
 12. Theparallel bioreactor system of claim 1, further comprising a perfusionsystem, wherein the perfusion system comprises a bracket having twoguide posts and a perfusion type culture bag vessel fixed between thetwo guide posts, and lifting adjustment buttons are arranged at lowerends of the guide posts.
 13. The parallel bioreactor system of claim 12,wherein the perfusion type culture bag vessel is connected with theculture vessel through pipelines and the connection holes of themultifunctional cover plate to form an outer circulation type perfusionculture mode.
 14. The parallel bioreactor system of claim 1, wherein thecontrol system monitors and controls one or more of the followingparameters in the disposable culture bag disposed in the culture vessel:liquid level, temperature, pH value and dissolved oxygen level.
 15. Theparallel bioreactor system of claim 1, wherein the control system canindependently monitor and control each disposable culture bag.
 16. Theparallel bioreactor system of claim 15, wherein the control systemcomprises a master control console and a plurality of reactioncontrollers; the master control console controls the oscillator and isconnected to the plurality of reaction controllers to receive data fromthe plurality of reaction controllers and send a control instruction tothe plurality of reaction controllers; and each reaction controller isconnected to the corresponding culture vessel to receive the parametersfrom the culture vessel and carry out related operations on the culturevessel.
 17. The parallel bioreactor system of claim 15, wherein theplurality of culture vessels are divided into at least two groups,identical or different cells or microorganisms are cultured in eachgroup of culture vessels, and the control system controls the groups ofculture vessels to have different culture parameters therebetween.
 18. Aculture method for culturing cells and/or microorganisms by using theparallel bioreactor system of claim 1, comprising: independentlymonitoring and controlling each culture vessel through the controlsystem.
 19. The culture method of claim 18, comprising: dividing theplurality of culture vessels into at least two groups, culturingidentical or different cells or microorganisms in each group of culturevessels, and controlling the groups of culture vessels to have differentculture parameters therebetween.